RESEARCH ARTICLE Extensive Chromosomal Reorganization in the Evolution of New World Muroid Rodents (Cricetidae, Sigmodontinae): Searching for Ancestral Phylogenetic Traits Adenilson Leão Pereira1, Stella Miranda Malcher1, Cleusa Yoshiko Nagamachi1,2, Patricia Caroline Mary O’Brien3, Malcolm Andrew Ferguson-Smith3, Ana Cristina Mendes- Oliveira4, Julio Cesar Pieczarka1,2* 1 Laboratório de Citogenética, Centro de Estudos Avançados da Biodiversidade, ICB, Universidade Federal do Pará, Belém, Pará, Brasil, 2 CNPq Researcher, Brasília, Brasil, 3 Cambridge Resource Center for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom, 4 Laboratório de Zoologia e Ecologia de Vertebrados, ICB, Universidade Federal do Pará, Belém, Pará, Brasil * [email protected] OPEN ACCESS Citation: Pereira AL, Malcher SM, Nagamachi CY, O’Brien PCM, Ferguson-Smith MA, Mendes-Oliveira Abstract AC, et al. (2016) Extensive Chromosomal Reorganization in the Evolution of New World Muroid Sigmodontinae rodents show great diversity and complexity in morphology and ecology. Rodents (Cricetidae, Sigmodontinae): Searching for This diversity is accompanied by extensive chromosome variation challenging attempts to Ancestral Phylogenetic Traits. PLoS ONE 11(1): reconstruct their ancestral genome. The species Hylaeamys megacephalus–HME (Oryzo- e0146179. doi:10.1371/journal.pone.0146179 myini, 2n = 54), Necromys lasiurus—NLA (Akodontini, 2n = 34) and Akodon sp.–ASP (Ako- Editor: Riccardo Castiglia, Universita degli Studi di dontini, 2n = 10) have extreme diploid numbers that make it difficult to understand the Roma La Sapienza, ITALY rearrangements that are responsible for such differences. In this study we analyzed these Received: June 5, 2015 changes using whole chromosome probes of HME in cross-species painting of NLA and Accepted: December 13, 2015 ASP to construct chromosome homology maps that reveal the rearrangements between Published: January 22, 2016 species. We include data from the literature for other Sigmodontinae previously studied with probes from HME and Mus musculus (MMU) probes. We also use the HME probes Copyright: © 2016 Pereira et al. This is an open access article distributed under the terms of the on MMU chromosomes for the comparative analysis of NLA with other species already Creative Commons Attribution License, which permits mapped by MMU probes. Our results show that NLA and ASP have highly rearranged kar- unrestricted use, distribution, and reproduction in any yotypes when compared to HME. Eleven HME syntenic blocks are shared among the spe- medium, provided the original author and source are credited. cies studied here. Four syntenies may be ancestral to Akodontini (HME2/18, 3/25, 18/25 and 4/11/16) and eight to Sigmodontinae (HME26, 1/12, 6/21, 7/9, 5/17, 11/16, 20/13 and Data Availability Statement: Data from this study were uploaded as supplementary files. 19/14/19). Using MMU data we identified six associations shared among rodents from seven subfamilies, where MMU3/18 and MMU8/13 are phylogenetic signatures of Sigmo- Funding: This work was supported by Proc 552032/ 2010-7, Conselho Nacional de Desenvolvimento dontinae. We suggest that the associations MMU2entire, MMU6proximal/12entire, MMU3/ Científico e Tecnológico (CNPq) (http://www.cnpq.br; 18, MMU8/13, MMU1/17, MMU10/17, MMU12/17, MMU5/16, MMU5/6 and MMU7/19 are ICAAF 007/2011); Fundação de Amparo à Pesquisa part of the ancestral Sigmodontinae genome. do Estado do Pará (FAPESPA) (http://www.fapespa. pa.gov.br); Proc 2010/110447, Fundação de Amparo à Pesquisa do Estado do Pará (FAPESPA) (http:// www.fapespa.pa.gov.br). S. M. Malcher is the recipient of a CAPES Doctor Scholarship in PLOS ONE | DOI:10.1371/journal.pone.0146179 January 22, 2016 1/15 The Genomic Organization of Sigmodontine Rodents Neuroscience and Cell Biology, and A. L. Pereira is Introduction the recipient of a CAPES Doctor Scholarship in Genetics and Molecular Biology. The funders had no Muroids are the most diverse group of extant rodents, with approximately 1500 species distrib- role in study design, data collection and analysis, uted in six families [1]. The families Cricetidae and Muridae are the most species rich [1–3]. decision to publish, or preparation of the manuscript. The family Cricetidae is composed of six subfamilies [1]. Sigmodontinae comprises Competing Interests: The authors have declared approximately 400 species with the tribes Akodontini, Abothrichini, Ichthyomyini, Oryzo- that no competing interests exist. myini, Phyllotini, Reithrodontini, Sigmodontini, Thomasomyini, Wiedomyini and Euneo- myini, and 381 of these species are present in South America [1, 4–7]. Recent phylogenetic studies based on molecular data recognize this subfamily and its ten tribes as a monophyletic group [3, 7–10]. Two of these tribes are noteworthy for their taxonomic complexity, diversity and number of species. The most specie-rich tribe is Oryzomyini, with 118 species in 30 genera, and a distribution in rainforests to semi-arid regions of the Neotropical and Nearctic (southeastern section) regions [5, 11–13]. Akodontini is the second most speciose tribe, with 85 species in 15 genera, mainly in the tropical and sub-tropical forests of South America [1, 5]. G-banding is useful for the accurate identification of chromosomal homologies in karyo- types with few rearrangements, but is not useful in highly rearranged karyotypes, which makes it difficult to understand species with extensive chromosomal variation. Sigmodontinae have diploid numbers ranging from only 9–10 in species of genus Akodon to 92 in Neusticomys fer- reirai, Anotomys leander and Ichthyomys pittieri [1, 14–16]. This large variation is problematic when trying to identify the chromosomal rearrangements between the extreme karyotypes in Sigmodontinae. However, chromosome painting has been very successful in demonstrating such rearrangements. This has been shown in Akodon species with diploid numbers varying from 10 to 44 by Ventura et al. [17], in Akodon and Thaptomys by Suarez et al [18] and by Swier et al [19]inSigmodon genomes, which are quite stable, with few or no chromosome rear- rangements. Nagamachi et al. [20] have used the same strategy to demonstrate that the Oryzo- myini Hylaeamys megacephalus (2n = 54) and Cerradomys langguthi (2n = 46) are also highly rearranged. In addition, mouse whole chromosome probes were used to compare the karyo- types of the six Sigmodontinae species (five Akodontini and one Oryzomyini), and this enabled the reconstruction of chromosomal phylogeny and phylogenetic relationships [21–22]. How- ever, not all segments had their homeology detected in some genomes (e.g.: Necromys lasiurus, Thaptomys nigrita, Oligoryzomys flavescens, Akodon cursor, A. montensis, A. paranaensis and A. serrensis;[21–22]). Recently Di-Nizo et al. [23] using whole chromosome probes of the Oli- goryzomys moojeni (2n = 70), demonstrated that five species of the genus Oligoryzomys (Ory- zomyini) have a high degree of chromosomal reorganization; not all existing homeologous were detected. The use of probes from different species, and the gaps left by these studies, make it difficult to comprehend all the mechanisms involved in the reconstruction of the ancestral Sigmodontinae karyotype (See [24]). In this study, we constructed chromosomal homology maps between Akodontini Akodon sp. (2n = 10) and Necromys lasiurus (2n = 34) using cross species chromosome painting with Oryzomyini chromosomal probes from Hylaeamys megacephalus (2n = 54) to assess the mechanisms leading to the abrupt evolutionary rearrangements between species. We also compared our findings with those from the literature for species already mapped with H. megacephalus probes. Finally, we were able to compare our results on NLA using HME probes with some published results on NLA that used MMU probes. This allowed the identification of some corresponding regions of chromosome homology in studies made by different investi- gators using different probes. Our results reveal new findings for this important group of rodents and indicate new paths towards the reconstruction of the putative ancestral Sigmo- dontinae karyotype. PLOS ONE | DOI:10.1371/journal.pone.0146179 January 22, 2016 2/15 The Genomic Organization of Sigmodontine Rodents Material and Methods Ethics Statement JCP has a permanent field permit, number 13248 from “Instituto Chico Mendes de Conserva- ção da Biodiversidade”. The Cytogenetics Laboratory from UFPa has a special permit number 19/2003 from the Ministry of Environment for the transport of samples and permit 52/2003 for using the samples in research. The Ethics Committee (Comitê de Ética Animal da Universi- dade Federal do Pará) approved this research. The specimens were captured using a live cap- ture method designed for small mammals (traps type Sherman, Tomahawk and pitfalls [25]). Specimens were maintained in the lab with food and water, free from stress, until their eutha- nasia, made with the IP injection of barbiturates after local anesthetic (Ketamine HCl in combi- nation with Diazepam). Specimen characteristics and Chromosome preparations The specimens Necromys lasiurus (NLA, two males and one female) and Akodon sp. (ASP, one female and two males) were collected from the municipality of Parauapebas, Pará State, north- ern Brazil (Table 1). The sample was collected between October 2009 and January 2010. The identification of the specimens was made on the characteristics of skull and skin, and the voucher